VOLUME 118, NUMBER 51 DECEMBER 25, 1996 © Copyright 1996 by the American Chemical Society
Exploiting Hydration Hysteresis for High Activity of Cross-Linked Subtilisin Crystals in Acetonitrile Johann Partridge, Gillian A. Hutcheon, Barry D. Moore,* and Peter J. Halling Contribution from the Department of Bioscience and Biotechnology and the Department of Pure and Applied Chemistry, UniVersity of Strathclyde, Glasgow G1 1XW, U.K. ReceiVed April 26, 1996X
Abstract: The catalytic behavior of cross-linked subtilisin crystals in acetonitrile exhibits pronounced hysteresis. The transesterification activity of the crystals in anhydrous solvent varies as much as 80-fold, depending on the hydration history of the enzyme. In a comparison of drying methods, the highest rates of reaction were obtained with crystals dried by washing with organic solvent. Crystals dried over molecular sieves in air or solvent showed significantly lower activity. In all cases, full activity could be recovered in aqueous buffer. Evidence for hysteresis in hydration was obtained using a 2H-NMR method. Crystals rinsed with anhydrous solvent were found to retain approximately 70 waters per enzyme molecule more than those dried over molecular sieves. When different solvents were used to dry the enzyme crystals, the catalytic rate in anhydrous acetonitrile was found to vary significantlysmethanolwashed CLEC giving the lowest rates. The transesterification activity of the solvent-washed enzyme was found to be profoundly effected by the concentration of water in the system. Optimum conditions for ester production were obtained in anhydrous acetonitrile. Under these conditions, despite a lower initial rate, absence of the hydrolysis byproduct which inhibits the enzyme led to better synthetic yields.
Introduction There is considerable current interest in the use of crosslinked enzyme crystals (CLECs) as biocatalysts in aqueous and organic media. As with enzymes immobilized on supports, CLECs offer advantages over the conventional freeze-dried preparations when used in organic solvents: access to individual enzyme molecules will be improved, while particle aggregation and diffusional limitation are considerably reduced. In addition, it has been reported recently that CLECs exhibit exceptional stability properties.1 They remain active after prolonged * Correspondence on manuscript: Barry D. Moore, Department of Pure and Applied Chemistry, University of Strathclyde, Glasgow G1 1XW, U.K. FAX: (+44) 141 552 5664. E-mail:
[email protected]. X Abstract published in AdVance ACS Abstracts, December 1, 1996. (1) (a) St. Clair, N. L.; Navia, M. A. J. Am. Chem. Soc. 1992, 114, 73147316. (b) Lalonde, J. J. Chim. Oggi 1995, 13, 31-35. (c) Persichetti, R. A.; St. Clair, N. L.; Griffith, J. P.; Navia, M. A.; Margolin, A. L. J. Am. Chem. Soc. 1995, 117, 2732-2737. (d) Khalaf, N.; Govardhan, C. P.; Lalonde, J. J.; Persichetti, R. A.; Wang, Y. F.; Margolin, A. L. J. Am. Chem. Soc. 1996, 118, 5494-5495.
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exposure to high temperatures, near anhydrous organic solvents, and aqueous-organic solvent mixtures. They are also highly stable against autolysis and exogenous protease degradation. These characteristics make them potentially very attractive as catalysts for use in organic reaction mixtures. However, as yet there is limited information available on the optimal conditions for their use in such systems. The method by which conventional lyophilized or supported enzyme is prepared for use in organic media often varies. In some cases the biocatalyst, which is essentially dry, is added directly to the organic solvent which contains a known amount of water.2 However, in other studies the enzyme and solvent are preequilibrated to a known thermodynamic water activity (aw),3,4 either together or separately. Initial studies on the effect of hydration on catalytic activity of subtilisin CLECs in (2) (a) Zaks, A.; Klibanov, A. M. J. Biol. Chem. 1988, 263, 3194-3201. (b) Zaks, A.; Klibanov, A. M. J. Biol. Chem. 1988, 263, 8017-8021. (c) van Erp, S. H. M.; Kamenskaya, E. O., Khmelnitsky, Y. L. Eur. J. Biochem. 1991, 202, 379-384.
© 1996 American Chemical Society
12874 J. Am. Chem. Soc., Vol. 118, No. 51, 1996 acetonitrile have been reported recently.5 The CLEC utilized was acetonitrile washed and subjected to no further equilibration. The authors demonstrated that the initial rate of transesterification increased when the system aw was increased from
